Allergy tests for dogs are usually based on qualitative determination of positivity or negativity for antigens or relative determination of IgE whose levels are expressed by a laboratory unit defined by the clinical laboratory, with no quantitative determination performed for antigen-specific IgE, which can serve as an absolute index of allergies. This is also true for allergy tests for humans. Known conventional arts for measuring IgE include antigen-specific 1-stage assay (Patent Document 1), a strip test for in vitro allergy diagnosis (Patent Document 2), an immunological test for dietary allergic substances (Patent Document 3), clinical utility of qualitative determination of IgE in humans (Non-patent Document 1), a method of relative quantitation of canine IgE whose levels are expressed by a laboratory unit (Non-patent Document 2), a method of producing a human IgE-producing hybridoma (Non-patent Document 3), an antigen-specific canine IgE monoclonal antibody and preparation method thereof (Patent Document 4, Non-patent Document 4), the high-affinity IgE receptor α subunit (FcεRIα) (Non-patent Document 5) and the like. Although these conventional arts have realized qualitative or relatively quantitative determination of IgE, no method of absolute quantitation of antigen-specific IgE has been realized. Quantitation of antigen-specific IgE cannot be realized unless the following problems are solved.
(1) Problems with the Unit of Indication
A quantitative test of an antigen-specific IgE has been established in laboratory-based settings for some antigens (Japanese cedar pollen antigen and the like), wherein a standard curve is generated using a serum of a dog shown by an intradermal reaction to have been sensitized to an antigen X as “a standard serum” on the presumption that the serum contains specific IgE against the antigen X (Non-patent Document 2). In this method, however, because the standard serum used is set at each laboratory, antigen-specific IgE assay values are indicated by Laboratory Unit/ml. Therefore, the unit of indication is distinctly set for each experiment at each laboratory; if different experimental systems or different laboratories are involved, it is unavoidable to use different standard sera even for the same assay system, so that measured values could not be compared as they were. Even for an antigen-specific IgE assay system within the same laboratory, any different antigen necessitates the use of serum sensitized with the antigen, so that it has been impossible to unify the unit of indication for the different antigens. Therefore, even using the same assay system, a comparison of the amount of antigen-specific IgE between different antigens has been impossible (even when the same Laboratory Unit/ml is used, a comparison between different antigens is impossible). These circumstances are nearly the same as those in humans (Non-patent Document 1).
(2) Problems with Purification of Antigen-Specific IgE
A method of purifying desired antigen-specific canine IgE from a serum of a sensitized dog is theoretically possible. Because the dog essentially has a large amount of total IgE, a purification step in two stages is required for this purpose. First, total IgE in the serum is purified using an IgE adsorption column, and then an antigen-specific IgE is purified using an object antigen-adsorbing column. However, although this method enables purification of total IgE, purification of the antigen-specific IgE poses a major problem concerning whether or not the antigen-specific IgE adsorbed to the column can be successfully eluted and extracted because IgE strongly binds to antigens. For this reason, there have been no reported cases where antigen-specific IgE was actually purified by the above-described method using a canine or human serum, though there has been a case of purification of total IgE (Patent Document 4).
(3) Problems with Preparation of Hybridomas that Produce Antigen-Specific Canine IgE
If an antigen-specific canine IgE-producing hybridoma cell line can be established, it will be no longer necessary to purify antigen-specific canine IgE from a serum. To this end, in case of dogs, it is indispensable to prepare an antigen-sensitized dog and fuse cells of lymph nodes or spleen thereof, or peripheral blood lymphocytes cultured with stimulation with the object antigen, with mouse myeloma cells. However, the probability of successful fusion by this method is extremely low; only one clone (canine IgE against nematodal antigen) has been prepared to date (Non-patent Document 4). Moreover, even if the cell fusion is successful, a period of 1 year or more is taken to sort the desired clone and establish it as a cell line, so that this approach is impractical when several tens of kinds of antigens are involved. In human cases, it is ethically difficult to prepare such a hybridoma.
(4) Problems with Preparation Cost
If the standard serum to be used in (1) is prepared using an experimental dog, much cost is taken, posing a major problem. For example, with the assumption of a rearing cost of 30000 yen/dog/month as calculated from a purchasing cost of 100000 to 200000 yen per dog and duration of preparation of sensitized serum of 2 months, preparation of the standard serum will take at least 160000 to 260000 yen per antigen. Therefore, establishing an antigen-specific IgE quantitative testing system for 50 antigens will cost 8000000 to 13000000 yen. Although sensitizing one dog with several kinds of antigens may be considered to reduce the cost, the problem of unwanted detection of the cross-reactivity among the different antigens cannot be solved. Additionally, although a large amount of 100 to 200 mL of serum can be collected from one sensitized dog at one time (equivalent to daily measurements for 300 years), such a large amount is unnecessary for performing the examination. Rather, considering the freezer space for long-term storage of the serum and the like, the cost performance of preparing a sensitized dog is much worse than a mouse. In human cases, it is ethically impossible to prepare a standard serum.